Identification and deletion of the extracellular polysaccharide slime pathway across the genus Xanthobacter
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Foods and commodities made by microbial bioproduction offer a sustainable alternative to those produced from our traditional petrochemical-based supply chains. Increasingly, Xanthobacter strains are used for bioproduction because of their unique metabolic properties (e.g., CO 2 and N 2 fixation) and wide feedstock range, including growth on agricultural products (e.g., fructose, sucrose) and electrically generated substrates (e.g., hydrogen gas, methanol, acetate, and formate). Many Xanthobacter strains, however, produce an extracellular polysaccharide (EPS) slime that makes plating, pelleting, and handling these cultures challenging and shuttles away resources from biomass and other biosynthetic pathways. While slime-free mutants of slime- producing Xanthobacter strains have been isolated, the causative mutations were unknown. Thus, slime-free mutants need to be painstakingly reisolated based on phenotypic properties in each academically and industrially relevant strain, unless we know the gene to target. Here, we identified the gene and underlying mutation that causes a slime-free phenotype in the X. autotrophicus mutant 7CSF. When we deleted this gene, xsbA , slime production was abolished in the wild-type strain. We showed that deleting xsbA homologs similarly abolished slime production in slime-producers across the genus Xanthobacter ( X. versatilis, X. autotrophicus, X. flavus, and X. tagetidis ). We also identified a Wzx/Wzy-dependent EPS pathway downstream of xsbA that is conserved across slime-producing Xanthobacter strains. Lastly, abolishing slime production improved colony formation and pelleting, with minimal impact on growth. We demonstrated that challenging phenotypic properties in Xanthobacter strains can be removed, improving their usability as a chassis for sustainable bioproduction.
Importance
Xanthobacter strains are promising as microbial platforms for bioproduction to more sustainably make our foods and commodities than current petrochemical-based methods. However, many of these strains produce a sugary slime that can make handling these strains difficult and that shuttles resources away from more desirable pathways. While slime-free mutants of Xanthobacter strains have been isolated before, the mutation that caused this change was unknown, and slime-free mutants would need to be reisolated each time. Here, we identify a gene, designated xsbA , that can be deleted in slime-producing Xanthobacter strains to remove their ability to make slime. Strains with the gene deleted become easier to handle and process, with minimal impact on growth. We also discovered additional genes involved in Xanthobacter slime production, which could be used to manipulate and engineer the properties of their slime for useful purposes.
